Method for treating inflammation

ABSTRACT

A method for treating IL-20 induced inflammation. An antagonist to IL-20 is administered to treat inflammation and associated diseases. The antagonist can be an antibody that binds to IL-20 or its receptor or a soluble receptor that binds to IL-20. Examples of such diseases are adult respiratory disease, psoriasis, eczema, contact dermatitis, atopic dermatitis, septic shock, multiple organ failure, inflammatory lung injury, bacterial pneumonia, inflammatory bowel disease, rheumatoid arthritis, asthma, ulcerative colitis and Crohn&#39;s disease.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/043,046, filed Mar. 5, 2008, which is a continuation application ofU.S. application Ser. No. 11/458,666, filed Jul. 19, 2006, which is acontinuation application of U.S. application Ser. No. 10/424,658, filedApr. 28, 2003, now U.S. Pat. No. 7,189,394, which is a divisionalapplication of U.S. application Ser. No. 09/746,359, filed Dec. 22,2000, now U.S. Pat. No. 6,610,286, which claims priority under 35 U.S.C.§ 119(e) to U.S. Provisional Patent Application. 60/171,969, filed Dec.23, 1999 and U.S. Provisional Application No. 60/213,341 filed Jun. 22,2000.

BACKGROUND OF THE INVENTION

The teachings of all of the references cited herein are incorporated intheir entirety herein by reference.

Inflammation normally is a localized, protective response to trauma ormicrobial invasion that destroys, dilutes, or walls-off the injuriousagent and the injured tissue. It is characterized in the acute form bythe classic signs of pain, heat, redness, swelling, and loss offunction. Microscopically, it involves a complex series of events,including dilation of arterioles, capillaries, and venules, withincreased permeability and blood flow, exudation of fluids, includingplasma proteins, and leukocyte migration into the area of inflammation.

Diseases characterized by inflammation are significant causes ofmorbidity and mortality in humans. Commonly, inflammation occurs as adefensive response to invasion of the host by foreign, particularlymicrobial, material. Responses to mechanical trauma, toxins, andneoplasia also may results in inflammatory reactions. The accumulationand subsequent activation of leukocytes are central events in thepathogenesis of most forms of inflammation. Deficiencies of inflammationcompromise the host. Excessive inflammation caused by abnormalrecognition of host tissue as foreign or prolongation of theinflammatory process may lead to inflammatory diseases as diverse asdiabetes, arteriosclerosis, cataracts, reperfusion injury, and cancer,to post-infectious syndromes such as in infectious meningitis, rheumaticfever, and to rheumatic diseases such as systemic lupus erythematosusand rheumatoid arthritis. The centrality of the inflammatory response inthese varied disease processes makes its regulation a major element inthe prevention control or cure of human disease.

An important cytokine in the inflammatory process is interleukin-8(IL-8). IL-8 is a chemokine. It was identified as an agonist forneutrophils on the basis of two effects, chemotaxis and the release ofgranule enzymes. IL-8 binds to two receptors on neutrophils. IL-8receptors are also found on monocytes, basophils, and eosinophils. Inhuman fibroblasts, cytomegalovirus has been shown to induce theexpression of IL-8 receptors and to replicate more rapidly when cellsare exposed to IL-8. IL-8 is a potent chemoattractant for neutrophils;and the early stages of periodontal disease are characterized by theinflux of neutrophils. IL-8 is a potent inducer of angiogenesis inseveral angiogenesis-dependent chronic inflammatory conditions,including rheumatoid arthritis, psoriasis, and idiopathic pulmonaryfibrosis. Additionally, IL-8 is an important source of angiogenicactivity in human lung cancer. Also, IL-8 expression correlates withexperimental metastatic activity of some melanoma cell lines. Thus, aneffective method to treat inflammatory diseases would be to administeran agent that would inhibit IL-8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS.: 1-8 are schematic representations of a representative number ofembodiments of the IL-20 soluble receptor.

DESCRIPTION OF THE INVENTION

The present invention fills this need by providing for a method fortreating inflammation in which IL-20 plays a role, comprisingadministering to a mammal in need of treatment of inflammation anantagonist to IL-20 or an antagonist to the IL-20 receptor. Theantagonist to IL-20 can be an antibody, antibody fragment orsingle-chain antibody that binds to IL-20, a soluble receptor that bindsto IL-20. The antagonist to the IL-20 receptor can be an antibody,antibody fragment, single-chain antibody or small molecule that binds tothe IL-20 receptor. Also an anti-sense nucleotide that binds to the mRNAthat encodes IL-20 can be used as an antagonist. We have shown thatIL-20 up-regulates IL-8. Inflammatory diseases in which IL-8 plays asignificant role, and for which a decrease in IL-8 would be beneficialare, adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as asthma or bronchitis,bacterial pneumonia, psoriasis and inflammatory bowel disease such asulcerative colitis and Crohn's disease, eczema, atopic dermatitis andcontact dermatitis. Thus, antagonists to IL-20 can be used to treatthese diseases.

The teachings of all the references cited herein are incorporated intheir entirety by reference.

DEFINITIONS

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide. For example, representative contigs tothe polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are 5′-TAGCTTgagtct-3′and 3′-gtcgacTACCGA-5′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78(1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nucleotides in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear, monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

Introduction

The present invention relates to the treatment of inflammation andinflammatory diseases in which IL-20 plays a role either in initiatingor spreading or maintenance by administering to the afflicted individualan antagonist to IL-20. That antagonist can be an antibody to IL-20, asoluble receptor that binds to IL-20, an anti-sense molecule or anantibody that binds to either the IL-20RA subunit or the IL-20RB subunitof the IL-20 receptor.

IL-20 is defined and methods for producing it and antibodies to IL-20are contained in International Patent Application No. PCT/US98/25228,publication no. WO 99/27103, published Nov. 25, 1998 and U.S. patentapplication Ser. No. 09/313,458 filed May 17, 1999. The polynucleotideand polypeptide of human IL-20 are represented by SEQ ID NOs: 1-4, andmouse IL-20 by SEQ ID NOs: 5-9.

The receptor to IL-20 has been discovered and is a heterodimer comprisedof the polypeptide termed ‘IL-20RA’ (formally called Zcytor7) and apolypeptide termed ‘IL-20RB’. The IL-20RA polypeptide, nucleic acid thatencodes it, antibodies to IL-20RA, and methods for producing it aredisclosed in U.S. Pat. No. 5,945,511 issued Aug. 31, 1999. SEQ ID NOs:10-12 are the IL-20RA polynucleotides and polypeptides. Theextracellular domain of the human IL-20RA is comprised of a polypeptideselected from the group consisting of SEQ ID NOs: 12, 55, 63 and 65, thefull-length receptor subunit being comprised of SEQ ID NO: 11. Theextracellular domain of mouse IL-20RA is SEQ ID NO: 38, SEQ ID NO: 37being the entire mouse IL-20RA.

The extracellular domain of IL-20RB (SEQ ID NOs: 13-14, and a variantSEQ ID NOs: 18 and 19) is comprised of a polypeptide selected from thegroup consisting of SEQ ID NOs: 15, 59, 61, 67, 68 and 69. Preferably,the extracellular domain of the IL-20RA polypeptide and theextracellular domain of the IL-20RB polypeptide are covalently linkedtogether. In a preferred embodiment one extracellular subunitpolypeptide has a constant region of a heavy chain of an immunoglobulinfused to its carboxy terminus and the other extracellular subunit has aconstant light chain of an immunoglobulin (Ig) fused to its carboxyterminus such that the two polypeptides come together to form a solublereceptor and a disulfide bond is formed between the heavy and the lightIg chains. In another method, a peptide linker could be fused to the twocarboxy-termini of the polypeptides to form a covalently bonded solublereceptor.

SEQ ID NOs: 22 and 23 are constructs of the extracellular domain ofIL-20RA fused to a mutated human immunoglobulin gamma 1 constant regionproduced according to the procedure set forth in example 5. SEQ ID NO:62 is the predicted mature sequence without the signal sequence. SEQ IDNOs: 20 and 21 are constructs of the extracellular domain of IL-20RBfused to wild type human immunoglobulin kappa light chain constantregion produced according to the procedure of example 5. SEQ ID NO: 60is the predicted mature sequence without the signal sequence. FIG. 1depicts the heterotetramer produced by example 5.

SEQ ID NOs: 52 and 53 are constructs of the extracellular domain ofIL-20RA fused to a mutated human immunoglobulin gamma 1 constant regionproduced according to the procedure set forth in example 12. SEQ ID NO:54 is the predicted mature sequence without the signal sequence. SEQ IDNOs: 56 and 57 are constructs of the extracellular domain of IL-20RBfused to wild type human immunoglobulin kappa light chain constantregion produced according to the procedure of example 12. SEQ ID NO: 58is the predicted mature sequence without the signal sequence. Theresultant heterotetramer is almost identical to that produced by example5, the primary difference being the absence of a polypeptide linkerbetween the extracellular domains and the beginning of the Ig constantregions, 22 in FIG. 1. Hereinafter, the term “extracellular domain of areceptor” means the extracellular domain of the receptor or a portion ofthe extracellular domain that is necessary for binding to its ligand, inthis case the ligand being IL-20.

One can link together the extracellular domains of IL-20RA and IL-20RBin a number of ways such that the resultant soluble receptor can bind toIL-20. FIGS. 1-8 illustrate a representative number of embodiments ofthe present invention. Common elements in each of the drawings are giventhe same number. FIG. 1 represents the embodiment of the presentinvention produced according to example 5 below. The soluble receptorconstruct, designated 10, is comprised of two IL-20 binding sitepolypeptide chains designated 12 and 14. Each binding site is comprisedof the extracellular domain of IL-20RA, designated 16, and theextracellular domain of IL-20RB designated 18.

The extracellular domain, 16, of IL-20RA is linked to the constant heavyone (CH1) domain, 20, of the human immunoglobulin gamma 1 heavy chainconstant region via linker 22, which is SEQ ID NO:72. The CH1 domain,20, is then linked to the CH2 domain, 24, via hinge region 23. The CH2domain, 24, is linked to the CH3 domain, 26, via hinge region 25.

Comparing the construct of FIG. 1 with SEQ ID NO:22, the extracellulardomain, 16, of IL-20RA extends from amino acid residues 36, a valine, toand including amino acid residue 249, a glutamine of SEQ ID NO:22.Polypeptide linker, 22, extends from amino acid residue 250, a glycineto and including amino acid residue 264, a serine, of SEQ ID NO:22. TheCH1 domain, 22 of FIG. 1, extends from amino acid residue 265, analanine, to and including amino acid residue 362, a valine, of SEQ IDNO:22. Hinge region 23 of FIG. 1 extends from amino acid residue 363, aglutamic acid to and including amino acid residue 377, a proline, of SEQID NO: 22. Chains 12 and 14 are disulfide-bonded together by means ofdisulfide bonds 28 and 30. The disulfide bonds are formed between theheavy chains by the cysteine residues at positions 373 and 376 of SEQ IDNO: 22 of each of the two heavy chains.

Extracellular domain, 18, of IL-20RB is linked to the constant region ofthe human kappa light chain (CL), 34 of FIG. 1 via polypeptide linker32, which is the polypeptide SEQ ID NO: 72. The extracellular domain,18, of IL-20RB extends from amino acid residue 30, a valine, to andincluding amino acid residue 230, an alanine, of SEQ ID NO: 20.Polypeptide linker, 32, extends from amino acid residue 231, a glycine,to and including amino acid residue 245, a serine, of SEQ ID NO:20. Thekappa constant light region, 34, extends from amino acid residue 246, anarginine, to and including the final amino acid residue 352, a cysteine,of SEQ ID NO:20. The cysteine at position 352 of SEQ ID NO: 20 forms adisulfide bond, 36 in FIG. 1, with the cysteine at position 367 of SEQID NO: 22. The constant light chain 34 is thus linked to the hingeregion, 23, by disulfide bond, 36. In this way, the extracellulardomain, 16, of IL-20RA is linked to the extracellular domain, 18, ofIL-20RB to form a soluble receptor.

If the cysteine residues at positions 373 and 376 of SEQ ID NO:22 werechanged to different amino acid residues, the two IL-20 bindingpolypeptides, 12 and 14, would not be disulfide bonded together andwould form a construct shown in FIG. 2. having hinge region, 27.

FIG. 3 shows a very simple soluble receptor 38 of the present inventionwherein extracellular domain, 16, of IL-20RA is connected to theextracellular domain, 18, of IL-20RB by means of a polypeptide linker,40. The polypeptide linker extends from the amino terminus ofextracellular domain, 16, of IL-20RA and is connected to the carboxylterminus of the extracellular domain, 18, of IL-20RB. The polypeptidelinker should be between 100-240 amino acids in length, preferably about170 amino acid residues in length. A suitable linker would be comprisedof glycine and serine residues. A possible linker would be multipleunits of SEQ ID NO: 72, preferably about 12.

FIG. 4 shows an embodiment that has the extracellular domain, 16, ofIL-20RA linked to the extracellular domain, 18, of IL-20RB by means oflinker 40, as in FIG. 3. While the extracellular domain, 16, of IL-20RAis linked to the CH1 domain, 20, as in FIG. 1 by means of polypeptidelinker 42, which should be about 30 amino acid residues in length. Anideal linker would be comprised of glycine and serine as in SEQ ID NO:72, and the hinge sequence, 23 of FIG. 1.

FIG. 5 shows another possible embodiment of the present invention. Inthis embodiment, a polypeptide linker 44 of about 15 amino acid residue,e.g. SEQ ID NO: 72, links the carboxyl terminus of the extracellulardomain, 18, of IL-20RB with the amino terminus of the extracellulardomain, 16, of IL-20RA. A polypeptide linker 46 of about 30 amino acidresidues extends from the carboxy terminus of the extracellular domain,16, of IL-20RA to the CH2 domain. The carboxyl terminus of linker 46would preferably be comprised of the hinge region extending from aminoacid residue 363, a glutamic acid to and including amino acid residue377, a proline, of SEQ ID NO: 22. Nonetheless, polypeptide linker 46would ideally have at least one cysteine residue at its carboxylterminus so a disulfide bond could be formed.

The soluble IL-20 receptor of FIG. 6 is identical to that of FIG. 1except for the CH3 domain, 26 of FIG. 1, is not present on theembodiment of FIG. 6. The CH3 region begins at amino acid residue 488, aglycine, and extends to the last residue 594 of SEQ ID NO: 22.

FIG. 7 shows a soluble IL-20 receptor construct that is identical to theconstruct of FIG. 1 except both the CH2, and CH3 domains are absent. TheCH2 and CH3 domains run from amino acid residue 378, an alanine, to theend of the polypeptide sequence of SEQ ID NO: 22.

FIG. 8 shows a construct wherein both IL-20RA, 16, and IL-20RB have apolypeptide linker, 48, fused to their respective carboxyl termini. Eachpolypeptide linker has two cysteine residues such that when they areexpressed the cysteines form two disulfide bonds, 50 and 52. In thiscase the polypeptide linker is comprised of the hinge region, 23 inFIG. 1. The hinge region is comprised of amino acid residues 363, aglutamine, to and including amino acid residue 377 of SEQ ID NO: 22.

In another aspect of the invention, a method is provided for producing asoluble receptor comprised of extracellular domains of IL-20RA andIL-20RB comprising (a) introducing into a host cell a first DNA sequencecomprised of a transcriptional promoter operatively linked to a firstsecretory signal sequence followed downstream by and in proper readingframe the DNA that encodes the extracellular portion of IL-20RA and theDNA that encodes an immunoglobulin light chain constant region; (b)introducing into the host cell a second DNA construct comprised of atranscriptional promoter operatively linked to a second secretory signalfollowed downstream by and in proper reading frame a DNA sequence thatencodes the extracellular portion of IL-20RB and a DNA sequence thatencodes an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4; (c)growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a fusion proteincomprised of the extracellular domain of IL-20RA and IL-20RB; and (d)isolating the polypeptide from the host cell. In one embodiment, thesecond DNA sequence further encodes an immunoglobulin heavy chain hingeregion wherein the hinge region is joined to the heavy chain constantregion domain. In another embodiment, the second DNA sequence furtherencodes an immunoglobulin variable region joined upstream of and inproper reading frame with the immunoglobulin heavy chain constantregion.

In an alternative embodiment, a method is provided for producing asoluble receptor comprised of the extracellular domains of IL-20RA andIL-20RB comprising (a) introducing into a host cell a first DNA sequencecomprised of a transcriptional promoter operatively linked to a firstsecretory signal sequence followed downstream by and in proper readingframe the DNA that encodes the extracellular portion of IL-20RB and theDNA that encodes an immunoglobulin light chain constant region; (b)introducing into the host cell a second DNA construct comprised of atranscriptional promoter operatively linked to a second secretory signalfollowed downstream by and in proper reading frame a DNA sequence thatencodes the extracellular portion of IL-20RA and a DNA sequence thatencodes an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4; (c)growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a dimerizedheterodimeric fusion protein comprised of the extracellular domain ofIL-20RA and IL-20RB; and (d) isolating the dimerized polypeptide fromthe host cell. In one embodiment, the second DNA sequence furtherencodes an immunoglobulin heavy chain hinge region wherein the hingeregion is joined to the heavy chain constant region domain. In anotherembodiment, the second DNA sequence further encodes an immunoglobulinvariable region joined upstream of and in proper reading frame with theimmunoglobulin heavy chain constant region. (See U.S. Pat. No.5,843,725.)

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. The binding affinity of an antibodycan be readily determined by one of ordinary skill in the art, forexample, by Scatchard analysis. A variety of assays known to thoseskilled in the art can be utilized to detect antibodies that bind toprotein or peptide. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold SpringHarbor Laboratory Press, 1988). Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay.

We have shown that IL-20 up-regulates IL-8. Inflammatory diseases inwhich IL-8 plays a significant role, and for which a decrease in IL-8would be beneficial are, adult respiratory disease (ARD), septic shock,multiple organ failure, inflammatory lung injury such as asthma orbronchitis, bacterial pneumonia, psoriasis and inflammatory boweldisease such as ulcerative colitis and Crohn's disease, eczema, atopicdermatitis and contact dermatitis. Thus, antagonists to IL-20 can beused to treat these diseases.

Biology of IL-20, its Receptor and its Role in Psoriasis

Two orphan class TI cytokine receptors, both of which are expressed inskin, were identified as IL-20 receptor subunits. Both IL-20 receptorsubunits are required for ligand binding, distinguishing their role fromthat of subunits in the four other known class II cytokine receptors.IL-20RA and IL-20RB are also coexpressed in a number of human tissuesbesides skin, including ovary, adrenal gland, testis, salivary gland,muscle, lung, kidney, heart and to a lesser degree the small intestinesuggesting additional target tissues for IL-20 action. Additionally, wehave detected IL-20RA mRNA but not IL-20RB mRNA in several human tissuesincluding stomach, thyroid, pancreas, uterus, brain and prostatesuggesting that IL-20RA may partner with other class II receptorsubunits. We conclude that the IL-20 heterodimeric receptor isstructurally similar to other class II cytokine receptors and isexpressed in skin where we have demonstrated activity of the IL-20ligand.

Two lines of evidence indicate that a role IL-20 and its receptor areinvolved in psoriasis. This multigenic skin disease is characterized byincreased keratinocyte proliferation, altered keratinocytedifferentiation, and infiltration of immune cells into the skin. Thefirst line of evidence for a role of IL-20 in psoriasis is that theobserved hyperkeratosis and thickened epidermis in the transgenic micethat resemble human psoriatic abnormalities. Decreased numbers oftonofilaments, thought to be related to defective keratinization, are astriking feature of human psoriasis. Intramitochondrial inclusions havebeen found in both chemically induced and naturally occurringhyperplastic skin conditions in mice. The cause of the inclusions andtheir effects on mitochondrial function, if any, are unknown. Weconclude that IL-20 transgenic mice exhibit many of the characteristicsobserved in human psoriasis.

A second line of evidence that implicates the IL-20 receptor inpsoriasis is that both IL-20RA and IL-20RB mRNA are markedly upregulatedin human psoriatic skin compared to normal skin. Both IL-20 receptorsubunits are expressed in keratinocytes throughout the epidermis and arealso expressed in a subset of immune and endothelial cells. We proposethat increased expression of an activated IL-20 receptor may alter theinteractions between endothelial cells, immune cells and keratinocytes,leading to dysregulation of keratinocyte proliferation anddifferentiation.

A crucial step in understanding the function of a novel cytokine is theidentification and characterization of its cognate receptor. We havesuccessfully used a structure-based approach to isolate a novelinterleukin that ultimately led to the isolation of its receptor. IL-20stimulates signal transduction in the human keratinocyte HaCaT cellline, supporting a direct action of this novel ligand in skin. Inaddition, IL-1β, EGF and TNF-α, proteins known to be active inkeratinocytes and to be involved with proliferative and pro-inflammatorysignals in skin, enhance the response to IL-20. In both HaCaT and BHKcells expressing the IL-20 receptor, IL-20 signals through STAT3.

Use of Antagonist to IL-20 to Treat Psoriasis

As indicated in the discussion above and the examples below, IL-20 isinvolved in the pathology of psoriasis. The present invention is inparticular a method for treating psoriasis by administering antagoniststo IL-20. The antagonists to IL-20 can either be a soluble receptor thatbinds to IL-20 or antibodies, single chain antibodies or fragments ofantibodies that bind to either IL-20 or the IL-20 receptor. Theantagonists will thus prevent activation of the IL-20 receptor.

Psoriasis is one of the most common dermatologic diseases, affecting upto 1 to 2 percent of the world's population. It is a chronicinflammatory skin disorder characterized by erythematous, sharplydemarcated papules and rounded plaques, covered by silvery micaceousscale. The skin lesions of psoriasis are variably pruritic. Traumatizedareas often develop lesions of psoriasis. Additionally, other externalfactors may exacerbate psoriasis including infections, stress, andmedications, e.g. lithium, beta blockers, and anti-malarials.

The most common variety of psoriasis is called plaque type. Patientswith plaque-type psoriasis will have stable, slowly growing plaques,which remain basically unchanged for long periods of time. The mostcommon areas for plaque psoriasis to occur are the elbows knees, glutealcleft, and the scalp. Involvement tends to be symmetrical. Inversepsoriasis affects the intertriginous regions including the axilla,groin, submammary region, and navel, and it also tends to affect thescalp, palms, and soles. The individual lesions are sharply demarcatedplaques but may be moist due to their location. Plaque-type psoriasisgenerally develops slowly and runs an indolent course. It rarelyspontaneously remits.

Eruptive psoriasis (guttate psoriasis) is most common in children andyoung adults. It develops acutely in individuals without psoriasis or inthose with chronic plaque psoriasis. Patients present with many smallerythematous, scaling papules, frequently after upper respiratory tractinfection with beta-hemolytic streptococci. Patients with psoriasis mayalso develop pustular lesions. These may be localized to the palms andsoles or may be generalized and associated with fever, malaise,diarrhea, and arthralgias.

About half of all patients with psoriasis have fingernail involvement,appearing as punctate pitting, nail thickening or subungualhyperkeratosis. About 5 to 10 percent of patients with psoriasis haveassociated joint complaints, and these are most often found in patientswith fingernail involvement. Although some have the coincidentoccurrence of classic Although some have the coincident occurrence ofclassic rheumatoid arthritis, many have joint disease that falls intoone of five type associated with psoriasis: (1) disease limited to asingle or a few small joints (70 percent of cases); (2) a seronegativerheumatoid arthritis-like disease; (3) involvement of the distalinterphalangeal joints; (4) severe destructive arthritis with thedevelopment of “arthritis mutilans”; and (5) disease limited to thespine.

Psoriasis can be treated by administering antagonists to IL-20. Thepreferred antagonists are either a soluble receptor to IL-20 orantibodies, antibody fragments or single chain antibodies that bind toeither the IL-20 receptor or to IL-20. The antagonists to IL-20 can beadministered alone or in combination with other established therapiessuch as lubricants, keratolytics, topical corticosteroids, topicalvitamin D derivatives, anthralin, systemic antimetabolites such asmethotrexate, psoralen-ultraviolet-light therapy (PUVA), etretinate,isotretinoin, cyclosporine, and the topical vitamin D3 derivativecalcipotriol. The antagonists, in particularly the soluble receptor orthe antibodies that bind to IL-20 or the IL-20 receptor can beadministered to individual subcutaneously, intravenously, ortransdermally using a cream or transdermal patch that contains theantagonist of IL-20. If administered subcutaneously, the antagonist canbe injected into one or more psoriatic plaques. If administeredtransdermally, the antagonists can be administered directly on theplaques using a cream containing the antagonist to IL-20.

Use of Antagonists to IL-20 to Treat Inflammatory Conditions of theLung.

Antagonists to IL-20 can be administered to a person who has asthma,bronchitis or cystic fibrosis or other inflammatory lung disease totreat the disease. The antagonists can be administered by any suitablemethod including intravenous, subcutaneous, bronchial lavage, and theuse of inhalant containing an antagonist to IL-20.

Administration of Antagonists to IL-20

The quantities of antagonists to IL-20 necessary for effective therapywill depend upon many different factors, including means ofadministration, target site, physiological state of the patient, andother medications administered. Thus, treatment dosages should betitrated to optimize safety and efficacy. Typically, dosages used invitro may provide useful guidance in the amounts useful for in vivoadministration of these reagents. Animal testing of effective doses fortreatment of particular disorders will provide further predictiveindication of human dosage. Methods for administration include oral,intravenous, peritoneal, intramuscular, transdermal or administrationinto the lung or trachea in spray form by means or a nebulizer oratomizer. Pharmaceutically acceptable carriers will include water,saline, buffers to name just a few. Dosage ranges would ordinarily beexpected from 1 μg to 1000 μg per kilogram of body weight per day. Adosage for an average adult of the IL-20 soluble receptor would be about25 mg given twice weekly as a subcutaneous injection. A dosage of anantibody that binds to IL-20 would be about 25 mg given twice weekly. Amixture of antibodies that bind to the IL-20RA and IL-20RB subunitswould be about 25 mg given twice weekly for an adult. Injections couldbe given at the site of psoriatic lesions for the treatment ofpsoriasis. For subcutaneous or intravenous administration of theantagonist to IL-20, the antibody or soluble receptor can be inphosphate buffered saline. Also in skin diseases such as psoriasis, theantagonist to IL-20 can be administered via an ointment or transdermalpatch. The doses by may be higher or lower as can be determined by amedical doctor with ordinary skill in the art. For a complete discussionof drug formulations and dosage ranges see Remington's PharmaceuticalSciences, 18^(th) Ed., (Mack Publishing Co., Easton, Pa., 1996), andGoodman and Gilman's: The Pharmacological Bases of Therapeutics, 9^(th)Ed. (Pergamon Press 1996).

The invention is further illustrated by the following non-limitingexamples.

Example 1 Up-Regulation of IL-8 by IL-20 Methods:

Normal Human Epidermal neonatal keratinocytes (NHEK) (from Clonetics) atpassage 2 were plated and grown to confluency in 12 well tissue cultureplates. KGM (Keratinocyte growth media) was purchased from Clonetics.When cells reached confluency, they were washed with KGM media minusgrowth factors=KBM (keratinocyte basal media). Cells were serum starvedin KBM for 72 hours prior to the addition of test compounds. Thrombin at1 I.U./mL and trypsin at 25 nM were used as positive controls. One mL ofmedia/well was added. KBM only was used as the negative control.

IL-20 was made up in KBM media and added at varying concentrations, from2.5 μg/ml down to 618 ng/mL in a first experiment and from 2.5 μg/mLdown to 3 ng/mL in a second experiment.

Cells were incubated at 37° C., 5% CO₂ for 48 hours. Supernatants wereremoved and frozen at −80° C. for several days prior to assaying forIL-8 and GM-CSF levels. Human IL-8 Immunoassay kit #D8050 (Rand DSystems, Inc.) and human GM-CSF Immunoassay kit #HSGMO (Rand D Systems,Inc.) were used to determine cytokine production followingmanufacturer's instructions.

Results

The results indicated that the expression of IL-8 and GM-CSF wereinduced by IL-20.

Example 2 Cloning of IL-20RB Cloning of IL-20RB Coding Region

Two PCR primers were designed based on the sequence from InternationalPatent Application No. PCT/US99/03735 (publication no. WO 99/46379)filed on Mar. 8, 1999. SEQ ID NO: 16 contains the ATG (Met1) codon withan EcoRI restriction site, SEQ ID NO: 17 contains the stop codon (TAG)with an XhoI restriction site. The PCR amplification was carried outusing a human keratinocyte (HaCaT) cDNA library DNA as a template andSEQ ID NO: 16 and SEQ ID NO: 17 as primers. The PCR reaction wasperformed as follows: incubation at 94° C. for 1 min followed by 30cycles of 94° C. for 30 sec and 68° C. for 2 min, after additional 68°C. for 4 min, the reaction was stored at 4° C. The PCR products were runon 1% Agarose gel, and a 1 kb DNA band was observed. The PCR productswere cut from the gel and the DNA was purified using a QIAquick GelExtraction Kit (Qiagen). The purified DNA was digested with EcoRI andXhoI, and cloned into a pZP vector that was called pZP7N. A pZP plasmidis a mammalian expression vector containing an expression cassettehaving the mouse metallothionein-1 promoter, human tPA leader peptide,multiple restriction sites for insertion of coding sequences, a Glu-Glutag, and a human growth hormone terminator. The plasmid also has an E.coli origin of replication, a mammalian selectable marker expressionunit having an SV40 promoter, an enhancer and an origin of replication,as well as a DHFR gene, and the SV40 terminator. Several IL-20RB-pZP7Nclones were sequenced. They all contain three non-conservative mutationscompared with the sequence of IL-20RB in PCT/US99/03735: (sequenceIL-20RB-pZP7N), 146 Pro (CCC)-Thr (ACC), 148 His (CAT)-Asp (GAT), and171 Thr (ACG)-Arg (AGG).

To verify the three substitutions in IL-20RB-pZP7N clone, PCRamplification was carried out using three difference cDNA sources—fetalskin marathon cDNA, HaCaT cDNA library DNA, and prostate smooth musclecDNA library DNA—as templates. The PCR products were gel purified andsequenced. The sequence of each of the three PCR products was consistentwith that of the IL-20RB-pZP7N clone. IL-20RB is SEQ ID NO: 13 and 14,and the mature extracellular domain is SEQ ID NO: 15.

Example 3 Binding of IL-20 to IL-20RB/IL-20RA Heterodimer

A cell-based binding assay was used to verify IL-20 binds toIL-20RA-IL-20RB heterodimer.

Expression vectors containing known and orphan Class II cytokinereceptors (including IL-20RA and IL-20RB) were transiently transfectedinto COS cells in various combinations, which were then assayed fortheir ability to bind biotin-labeled IL-20 protein. The results showIL-20RB-IL-20RA heterodimer is a receptor for IL-20. The procedure usedis described below.

The COS cell transfection was performed in a 12-well tissue cultureplate as follows: 0.5 μg DNA was mixed with medium containing 5 μllipofectamine in 92 μl serum free Dulbecco's modified Eagle's medium(DMEM) (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5mg insulin, 1 μg selenium and 5 mg fetuin in 500 ml DMEM), incubated atroom temperature for 30 minutes and then added to 400 μl serum free DMEMmedia. This 500 μl mixture was then added to 1.5×10⁵ COS cells/well andincubated for 5 hours at 37° C. 500 μl 20% fetal bovine serum (FBS) DMEMmedia was added and incubated overnight.

The assay, a modification of the “secretion trap” (Davis, S., et al.,Cell 87: 1161-1169 (1996), was performed as follows: cells were rinsedwith PBS/1% bovine serum albumin (BSA) and blocked for 1 hour with TNB(0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Blocking Reagent (NEN RenaissanceTSA-Direct Kit Cat #NEL701) in water). This was followed by a one-hourincubation with 3 μg/ml biotinylated IL-20 protein in TNB. Cells werewashed with PBS/1% BSA and incubated for another hour with 1:300 dilutedstreptavidin-HRP (NEN kit) in TNB. Following another wash, cells werefixed for 15 minutes with 1.8% Formaldehyde in phosphate-buffered saline(PBS). Cells were then washed with TNT (0.1 M Tris-HCL, 0.15 M NaCl, and0.05% Tween-20 in water). Positive binding signals were detectedfollowing a five-minute incubation with fluorescein tyramide reagentdiluted 1:50 in dilution buffer (NEN kit). Cells were washed with TNT,preserved with Vectashield Mounting Media (Vector Labs) diluted 1:5 inTNT, and visualized using an FITC filter on an inverted fluorescentmicroscope.

Example 4 Up-Regulation of Inflammatory Cytokines by IL-20 CellTreatment

The human keratinocyte cell line, HaCaT was grown at 37° C. to severaldays post-confluence in T-75 tissue culture flasks. At this point,normal growth media (DMEM+10% FBS) was removed and replaced withserum-free media. Cells were then incubated for two days at 37° C. DMEMwas then removed and four flasks of cells per treatment were treatedwith one of each of the following conditions for four hours at 37° C.:recombinant human (rh) IL-1 alpha at 5 ng/mL, rh IL-1 alpha at 20 ng/mL,rh IL-1 alpha at 5 ng/mL+IL-20 at 1 μg/mL, IL-20 at 1 μg/mL, or rh IL-10at 10 ng/mL.

RNA Isolation

Following cytokine treatment, media was removed and cells were lysedusing a guanidium thiocyanate solution. Total RNA was isolated from thecell lysate by an overnight spin on a cesium chloride gradient. Thefollowing day, the RNA pellet was resuspended in a TE/SDS solution andethanol precipitated. RNA was then quantitated using aspectrophotometer, followed by a DNase treatment as per Section V.B. ofClontech's Atlas™ cDNA Expression Arrays User Manual (versionPT3140-1/PR9X390, published Nov. 5, 1999). Quality of RNA samples wasverified by purity calculations based on spec readings, and byvisualization on agarose gel. Genomic contamination of the RNA sampleswas ruled out by PCR analysis of the beta-actin gene.

Probe Synthesis

Clontech's protocols for polyA+ enrichment, probe synthesis andhybridization to Atlas™ arrays were followed (see above, plus Atlas™Pure Total RNA Labeling System User Manual, PT3231-1/PR96157, publishedJun. 22, 1999). Briefly, polyA+RNA was isolated from 50 mg of total RNAusing streptavidin coated magnetic beads (by Clontech, Paolo Alto,Calif.) and a magnetic particle separator. PolyA+RNA was then labeledwith ^(alpha32)P-dATP via RT-PCR. Clontech CDS primers specific to the268 genes on the Atlas™ human cytokine/receptor array (Cat. #7744-1)were used in the reaction. Labeled probe was isolated using columnchromatography and counted in scintillation fluid.

Array Membrane Hybridization

Atlas™ arrays were pre-hybridized with Clontech ExpressHyb plus 100mg/mL heat denatured salmon sperm DNA for at least thirty minutes at 68°C. with continuous agitation. Membranes were then hybridized with1.9×10⁶ CPM/mL (a total of 1.14×10⁷ CPM) overnight at 68° C. withcontinuous agitation. The following day, membranes were washed forthirty minutes ×4 in 2×SSC, 1% SDS at 68° C., plus for thirty minutes ×1in 0.1×SSC, 0.5% SDS at 68° C., followed by one final room temperaturewash for five minutes in 2×SSC. Array membranes were then placed inKodak plastic pouches sealed and exposed to a phosphor imager screenovernight at room temperature. The next day, phosphor screens werescanned on a phosphor imager and analyzed using Clontech's AtlasImage™1.0 software.

Results Genes Up-Regulated by IL-20

-   1. Tumor necrosis factor (TNF) was up-regulated 1.9-2.4 fold by    IL-20.-   2. Placental growth factors 1 & 2 (PLGF) were up-regulated 1.9-2.0    fold by IL-20.-   3. Coagulating factor II receptor was up-regulated 2.0-2.5 fold by    IL-20.-   4. Calcitonin receptor was up-regulated 2.2-2.3 fold by IL-20.-   5. TNF-inducible hyaluronate-binding protein TSG-6 was up-regulated    2.1-2.2 fold by IL-20.-   6. Vascular endothelial growth factor (VEGF) receptor-1 precursor,    tyrosine-protein kinase receptor (FLT-1) (SFLT) was up-regulated    2.1-2.7 fold by IL-20.-   7. MRP-8 (calcium binding protein in macrophages MIF-related) was    up-regulated 2.9-4.1 fold by IL-20.-   8. MRP-14 (calcium binding protein in macrophages MIF-related) was    up-regulated 3.0-3.8 fold by IL-20.-   9. Relaxin H2 was up-regulated 3.14 fold by IL-20.-   10. Transforming growth factor beta (TGFβ) receptor III 300 kDa was    up-regulated 2.4-3.6 fold by IL-20.

Genes Showing Synergy with IL-20+IL-1 Treatment

-   1 Bone morphogenic protein 2a was up-regulated 1.8 fold with IL-20    treatment alone, 2.5 fold with IL-1 treatment alone, and 8.2 fold    with both IL-20 and IL-1 treatment together.-   2. MRP-8 was up-regulated 2.9 fold with IL-20 treatment alone, 10.7    fold with IL-1 treatment alone and 18.0 fold with both IL-20 and    IL-1 treatment together.-   3. Erythroid differentiation protein (EDF) was up-regulated 1.9 fold    with IL-20 treatment alone, 9.7 fold with IL-1 treatment alone and    19.0 fold with both IL-20 and IL-1 treatment together.-   4. MRP-14 (calcium binding protein in macrophages, MIF related) was    up-regulated 3.0 fold with IL-20 treatment alone, 12.2 fold with    IL-1 treatment alone and 20.3 fold with both IL-20 and IL-1    treatment together.-   5. Heparin-binding EGF-like growth factor was up-regulated 2.0 fold    with IL-20 treatment alone, 14 fold with IL-1 treatment alone and    25.0 fold with both IL-20 and IL-1 treatment together.-   6. Beta-thromboglobulin-like protein was up-regulated 1.5 fold with    IL-20 treatment alone, 15 fold with IL-1 treatment alone and 27 fold    with both IL-20 and IL-1 treatment together.-   7. Brain-derived neurotrophic factor (BDNF) was up-regulated 1.7    fold with IL-20 treatment alone, 25 fold with IL-1 treatment alone    and 48 fold with both IL-20 and IL-1 treatment together.-   8. Monocyte chemotactic and activating factor MCAF was up-regulated    1.3 fold with IL-20 treatment alone, 32 fold with IL-1 treatment    alone and 56 fold with both IL-20 and IL-1 treatment together.

Example 5 IL-20RA/RB Receptor-Ig Fusion Heterotetramer

The expression vector pEZE3 was used to express the recombinant IL-20receptor-Ig fusion protein. The plasmid pEZE3 is derived from pDC312.pDC312 was obtained through license from Immunex Corporation. Theplasmids pDC312 and pEZE3 contain an EASE segment as described in WO97/25420. The presence of the EASE segment in an expression vector canimprove expression of recombinant proteins two to eight fold in stablecell pools.

The plasmid pEZE3 is a tricistronic expression vector that may be usedto express up to three different proteins in mammalian cells, preferablyChinese Hamster Ovary (CHO) cells. The pEZE3 expression unit containsthe cytomegalovirus (CMV) enhancer/promoter, the adenovirus tripartiteleader sequence, a multiple cloning site for insertion of the codingregion for the first recombinant protein, the poliovirus type 2 internalribosome entry site, a second multiple cloning site for insertion of thecoding region for the second recombinant protein, anencephalomyocarditis virus internal ribosome entry site, a codingsegment for mouse dihydrofolate reductase, and the SV40 transcriptionterminator. In addition, pEZE3 contains an E. coli origin of replicationand the bacterial beta lactamase gene.

The IL-20 receptor-Ig fusion protein is a disulfide linkedheterotetramer consisting of two chains of the extracellular domain ofthe human IL-20RB fused to the wild type human immunoglobulin kappalight chain constant region and two chains of the human IL-20RA proteinextracellular domain fused to a mutated human immunoglobulin gamma 1constant region. The human immunoglobulin gamma 1 constant regioncontains amino acid substitutions to reduce FcγRI binding and C1qcomplement fixation.

The human IL-20RB extracellular domain human immunoglobulin kappa lightchain constant region fusion construct was generated by overlap PCR. TheIL-20RB coding segment consists of amino acids 1 to 230. The templateused for the PCR amplification of the IL-20R segment was generatedIL-20RB human kappa light chain constant region expression construct asdescribed below in Example 12. Oligonucleotide primers SEQ ID NO: 24 andSEQ ID NO: 25 were used to amplify the IL-20RB segment. The entire wildtype human immunoglobulin kappa light chain constant region was used.The template used for the PCR amplification of the wild type humanimmunoglobulin kappa light chain constant region segment was generatedIL-20RB human kappa light chain constant region expression construct asdescribed in Example 12. Oligonucleotide primers SEQ ID NO: 26 and SEQID NO: 27 were used to amplify the wild type human immunoglobulin kappalight chain constant region. The two protein coding domains were linkedby overlap PCR using oligonucleotides SEQ ID NO: 24 and SEQ ID NO: 27. A(Gly₄Ser)₃ (SEQ ID NO: 72) peptide linker was inserted between the twoprotein domains. The (Gly₄Ser)₃ peptide linker was encoded on the PCRprimers SEQ ID NO: 26 and SEQ ID NO:25. The resultant IL-20RBextracellular domain/kappa light chain constant region fusion constructis shown by SEQ ID NOs: 20 and 21. The predicted mature polypeptide,minus the signal sequence, is SEQ ID NO: 60. The portion of theextracellular domain of IL-20RB that was actually used was comprised ofthe amino acid sequence of SEQ ID NO: 61. N-terminal sequencing resultedin the predicted amino acid sequence.

The human IL-20RA extracellular domain human immunoglobulin gamma 1heavy chain constant region fusion construct was generated by overlapPCR of four separate DNA fragments, each generated by separate PCRamplification reactions. The first fragment contained an optimized tPA(tissue plasminogen activator) signal sequence. The tPA signal sequencewas amplified using oligonucleotide primers SEQ ID NO: 28 and SEQ ID NO:29 using an in-house previously generated expression vector as thetemplate. The second fragment contained the IL-20RA extracellulardomain-coding region consisting of amino acids 30 to 243 of SEQ ID NO:11. Oligonucleotide primers SEQ ID NO: 30 and SEQ ID NO: 31 were used toamplify this IL-20RA segment using a previously generated clone ofIL-20RA as the template.

The human gamma 1 heavy chain constant region was generated from 2segments. The first segment containing the C_(H)1 domain was amplifiedusing oligonucleotide primers SEQ ID NO: 32 and SEQ ID NO: 33 using aclone of the wild type human gamma 1 heavy chain constant region as thetemplate. The second segment containing the remaining hinge, C_(H)2, andC_(H)3 domains of the human immunoglobulin gamma 1 heavy chain constantregion was generated by PCR amplification using oligonucleotide primersSEQ ID NO: 34 and SEQ ID NO: 35. The template used for this PCRamplification was from a previously generated human gamma 1 Fc constructthat contained codons for amino acid substitutions to reduce FcγRIbinding and C1q complement fixation as described in Example 12.

The four protein coding domains were linked by overlap PCR usingoligonucleotides SEQ ID NO: 28 and SEQ ID NO: 35. A (Gly₄Ser)₃ peptidelinker was inserted between the IL-20RA and CH1 protein domains. The(Gly₄Ser)₃ peptide linker was encoded on the PCR primers SEQ ID NO: 32and SEQ ID NO: 31. The IL-20RA extracellular domain/domain humanimmunoglobulin gamma 1 heavy constant region fusion protein and DNAsequence are shown in SEQ ID NOs: 22 and 23. The predicted maturepolypeptide sequence, minus the signal sequence, is SEQ ID NO: 62. Theportion of extracellular domain of IL-20RA that was actually used wascomprised of SEQ ID NO: 63.

The IL-20RB extracellular domain human immunoglobulin kappa light chainconstant region fusion coding segment was cloned into the second MCSwhile the human IL-20RA extracellular domain human immunoglobulin gamma1 heavy chain constant region fusion coding segment was cloned into thefirst MCS of pEZE3. The plasmid was used to transfect CHO cells. Thecells were selected in medium without hypoxanthine or thymidine and thetransgene was amplified using methotrexate. The presence of protein wasassayed by Western blotting using anti human gamma 1 heavy chainconstant region and anti human kappa light chain antibodies. N-terminalsequencing revealed that the optimized tPA leader was not completelycleaved. The observed mass indicated that the first residue of thepolypeptide sequence to be pyroglutamic acid, and the N-terminalsequence appears to be pyroEEIHAELRRFRRVPCVSGG (SEQ ID NO: 64), theunderlined portion being remnants of the tPA leader.

Example 6 IL-20 Transgenic Phenotype

Both human and mouse IL-20 were overexpressed in transgenic mice using avariety of promoters. The liver-specific mouse albumin promoter,directing expression of human IL-20, was used initially in an attempt toachieve circulating levels of protein. Subsequent studies were conductedusing the keratin 14 (K14) promoter, which primarily targets expressionto the epidermis and other stratified squamous epithelia; the mousemetallothionein-1 promoter, which gives a broad expression pattern; andthe EμLCK promoter, which drives expression in cells of the lymphoidlineage. Similar results were obtained in all four cases, possiblybecause these promoters all give rise to circulating levels of IL-20.

In all cases, transgenic pups expressing the IL-20 transgene weresmaller than non-transgenic littermates, had a shiny appearance withtight, wrinkled skin and died within the first few days after birth.Pups had milk in their stomachs indicating that they were able tosuckle. These mice had swollen extremities, tail, nostril and mouthregions and had difficulty moving. In addition, the mice were frail,lacked visible adipose tissue and had delayed ear and toe development.Low expression levels in liver (less than 100 mRNA molecules/cell) weresufficient for both the neonatal lethality and skin abnormalities.Transgenic mice without a visible phenotype either did not express thetransgene, did not express it at detectable levels, or were mosaic.

Histologic analysis of the skin of the IL-20 transgenic mice showed athickened epidermis, hyperkeratosis and a compact stratum corneumcompared to non-transgenic littermates. Serocellular crusts (scabs) wereobserved occasionally. Electron microscopic (EM) analysis of skin fromtransgenic mice showed intramitochondrial lipoid inclusions, mottledkeratohyaline granules, and relatively few tonofilaments similar to thatobserved in human psoriatic skin and in mouse skin disease models. Inaddition, many of the transgenic mice had apoptotic thymic lymphocytes.No other abnormalities were detected by histopathological analysis.These histological and EM results support and extend the observed grossskin alterations.

Example 7 Specificity and Affinity of IL-20 for its Receptor

The specificity and affinity of IL-20 for its receptor was determinedusing BHK cells stably transfected with IL-20RA, IL-20RB or bothreceptor subunits. Binding assays using radiolabeled ligand demonstratedthat IL-20 bound to BHK transfectants expressing both IL-20RA andIL-20RB but not to untransfected cells nor to transfectants expressingeither receptor subunit alone. Binding of ¹²⁵I-labeled IL-20 waseliminated in the presence of 100-fold excess of unlabeled IL-20 but notwith 100-fold excess of the unrelated cytokine, IL-21. The bindingaffinity (kD) of IL-20 to the IL-20RA/IL-20RB heterodimeric receptor wasdetermined to be approximately 1.5 nM.

Example 8 IL-20 Receptor Activation

To determine if IL-20 binding leads to receptor activation, thefactor-dependent pre-B cell line BaF3 was co-transfected with IL-20RAand IL-20RB and treated with IL-20 at various concentrations. IL-20stimulated proliferation in a dose-dependent manner and gave adetectable signal at 1.1 μM, with a half maximal response at 3.4 μM. Wenote that the IL-20 concentration for the half maximal proliferativeresponse in BaF3 cells is 1000× lower than that for half maximal bindingaffinity in BHK cells. Possible explanations for this large differenceinclude the use of different cell lines, different receptor expressionlevels and different assay outputs. IL-20 also stimulated signaltransduction in the biologically relevant human keratinocyte cell lineHaCaT, which naturally expresses IL-20RA and IL-20RB. Therefore, IL-20binds and activates the heterodimeric IL-20RA/IL-20RB receptor atconcentrations expected for a cytokine. While the negative controlscontaining untransfected BaF3

Example 9 Expression Analysis of IL-20RA and IL-20RB

RT-PCR analysis was performed on a variety of human tissues to determinethe expression pattern of IL-20RA and IL-20RB. Both receptor subunitsare most highly expressed in skin and testis. The significant result isthat IL-20RA and IL-20RB are both expressed in skin, where they havebeen shown to mediate the IL-20-induced response. Both IL-20RA andIL-20RB are also both expressed in monocytes, lung, ovary, muscle,testis, adrenal gland, heart, salivary gland and placenta. IL-20RA isalso in brain, kidney, liver, colon, small intestine, stomach, thyroid,pancreas, uterus and prostate while IL-20RB is not.

Example 10 IL-20RA and IL-20Rb mRNA are Up-Regulated in Psoriasis

In situ hybridization was used to determine whether IL-20 receptorexpression is altered in psoriasis. Skin samples from four psoriasispatients and three unaffected patients were assayed with probes specificfor the two-receptor subunit mRNAs. All four psoriatic skin samples hadhigh levels of IL-20RA and IL-20RB mRNA in keratinocytes whereas normalskin samples did not have detectable levels of either receptor subunitmRNA. Positive signals in psoriatic skin were also observed inmononuclear immune cells and in endothelial cells in a subset ofvessels. Therefore, both IL-20RA and IL-20RB are expressed inkeratinocytes, immune cells and endothelial cells, the major cell typesthought to interact in psoriasis.

Example 11 Cloning of Mouse IL-20RA

A cross-species hybridization probe was generated which contained thefull-length cDNA fragment encoding human IL-20RA. A Southern blot ofmouse genomic DNA and Northern blots of mouse RNA were performed todemonstrate that the human IL-20RA cDNA could specifically hybridize tomouse sequences. The Northern blot results indicated that mouse IL-20RARNA was present in mouse embryo day 15 and 17 as well as heart, brain,lung, liver, kidney, testes, spleen, thymus, liver, stomach, and smallintestine.

The human IL-20RA full length DNA hybridization probe was used to screena mouse genomic library. The library, which was obtained from Clontech(Palo Alto, Calif.), was generated from an MboI partial digest of mousegenomic DNA and cloned into the BamHI site of Lambda bacteriophage EMBL3SP6/T7. Positive bacteriophage was plaque purified and bacteriophage DNAwas prepared using Promega's Wizard Lambda Preps DNA PurificationSystem. Two genomic restriction enzyme fragments, a 5.7 kb EcoRIfragment and an 8.0 kb Sac fragment, were generated from the positivebacteriophage and subcloned into pBluescript. DNA sequence analysisrevealed the presence of 3 exons from the mouse ortholog to humanIL-20RA.

PCR primers from the 5′ UTR, SEQ ID NO: 40, and 3′ UTR, SEQ ID NO: 41,were designed to generate a full-length mouse IL-20RA sequence by PCRamplification. Mouse embryo 15 day plus 17 day cDNA was used as thetemplate for the PCR amplification. PCR products were subcloned andsequenced for confirmation. The mouse sequences are SEQ ID NOs: 36 and37. The mature extracellular domain is comprised of SEQ ID NO: 38.

Example 12 Construction of an IL-20 Receptor Heterotetramer

A vector expressing a secreted hIL-20RA/hIL-20B heterodimer wasconstructed. In this construct, the extracellular domain of hIL-20RA wasfused to the heavy chain of IgG gamma 1 (IgGγ1), while the extracellularportion of IL-20RB was fused to human kappa light chain (human κ lightchain).

Construction of IgG Gamma 1 and Human κ Light Fusion Vectors

The heavy chain of IgGγ1 was cloned into the Zem229R mammalianexpression vector (ATCC deposit No. 69447) such that any extracellularportion of a receptor having a 5′ EcoRI and 3′ NheI site can be clonedin, resulting in an N-terminal extracellular domain-C-terminal IgGγ1fusion. The IgGγ1 fragment used in this construct was made by using PCRto isolate the IgGγ1 sequence from a Clontech human fetal liver cDNAlibrary as template. A PCR reaction using oligos SEQ ID NO: 42 and SEQID NO: 43 was run as follows: 40 cycles of 94° for 60 sec., 53° C. for60 sec., and 72° for 120 sec.; and 72° C. for 7 minutes. PCR productswere separated by agarose gel electrophoresis and purified using aQiaQuick™ (Qiagen Inc., Valencia, Calif.) gel extraction kit. Theisolated, 990 bp, DNA fragment was digested with MluI and EcoRI(Boerhinger-Mannheim), extracted with QiaQuick™ gel extraction kit andligated with oligos SEQ ID NO: 44 and SEQ ID NO: 45, which comprise anMluI/EcoRI linker, into Zem229R previously digested with MluI and EcoRIusing standard molecular biology techniques disclosed herein. Thisgeneric cloning vector was called Vector #76 hIgGgamma1 w/Ch1 #786Zem229R (Vector #76). The polynucleotide sequence of the extracellulardomain of hIL-20RA fused to the heavy chain of IgG gamma 1 is show inSEQ ID NO: 52 and the corresponding polypeptide sequence shown in SEQ IDNO: 53, the mature polypeptide, minus the signal sequence beingcomprised of SEQ ID NO: 54. The portion of the extracellular domain ofIL-20RA used was comprised of SEQ ID NO: 55.

The human κ light chain was cloned in the Zem228R mammalian expressionvector (ATCC deposit No. 69446) such that any extracellular portion of areceptor having a 5′ EcoRI site and a 3′ KpnI site can be cloned in,resulting in an N-terminal extracellular domain-C-terminal human κ lightchain fusion. The human κ light chain fragment used in this constructwas made by using PCR to isolate the human κ light chain sequence fromthe same Clontech hFetal Liver cDNA library used above. A PCR reactionwas run using oligos SEQ ID NO: 46 and SEQ ID NO: 47. PCR products wereseparated by agarose gel electrophoresis and purified using a QiaQuick™(Qiagen) gel extraction kit. The isolated, 315 bp, DNA fragment wasdigested with MluI and EcoRI (Boerhinger-Mannheim), extracted withQiaQuick™ gel extraction kit and ligated with the MluI/EcoRI linkerdescribed above, into Zem228R previously digested with MluI and EcoRIusing standard molecular biology techniques disclosed herein. Thisgeneric cloning vector was called Vector #77 hκlight #774 Zem228R(Vector #77). The polynucleotide sequence of the extracellular portionof IL-20RB fused to human kappa light chain is shown in SEQ ID NO: 56and the corresponding polypeptide sequence shown in SEQ ID NO: 57, themature polypeptide, minus the signal sequence, is comprised of SEQ IDNO: 58. The portion of the extracellular domain of IL-20RB actually usedwas comprised of SEQ ID NO: 59.

Insertion of hIL-20RA and IL-20Rb Extracellular Domains into FusionVector Constructs

Using the construction vectors above, a construct having human IL-20RAfused to IgGγ1 was made. This construction was done by using PCR toobtain human IL-20RA receptor from hIL-20RA/IgG Vector #102 with oligosSEQ ID NO: 48 and SEQ ID NO: 49 under conditions described as follows:30 cycles of 94° C. for 60 sec., 57° C. for 60 sec., and 72° C. for 120sec.; and 72° C. for 7 min. The resulting PCR product was digested withEcoRI and NheI, gel purified, as described herein, and ligated into apreviously EcoRI and NheI digested and band-purified Vector #76 (above).The resulting vector was sequenced to confirm that the human IL-20Rα/IgGgamma 1 fusion (hIL-20RA/Ch1 IgG) was correct. The hIL-20RA/Ch1 IgGgamma 1 #1825 Zem229R vector was called vector #195. The IL-20RA/Ch1IgGγ1 sequence thus obtained is depicted by SEQ ID NOs: 52 and 53.N-terminal sequencing indicated the presence of the predicted maturepolypeptide sequence of SEQ ID NO: 54.

A separate construct having IL-20RB fused to K light was alsoconstructed. The IL-20RB/human κ light chain construction was performedas above by PCRing from DR1/7N-4 with oligos SEQ ID NO: 50 and SEQ IDNO: 51, digesting the resulting band with EcoRI and KpnI and thenligating this product into a previously EcoRI and KpnI digested andband-purified Vec #77 (above). The resulting vector was sequenced toconfirm that the IL-20RB/human κ light chain fusion (IL-20RB/κlight) wascorrect. This IL-20RB//κlight construct is shown by SEQ ID NOs: 56 and57. N-terminal sequencing of the resultant polypeptide indicated thepresence of the predicted mature amino acid sequence comprised of SEQ IDNO: 58. SEQ ID NO:59 is the mature portion of the extracellular domainof IL-20RB used.

Co-Expression of the Human IL-20RA and Human IL-20RB Receptors

Approximately 16 μg of each of vectors #194 and #195, above, wereco-transfected into BHK-570 cells (ATCC No. CRL-10314) usingLipofectamine™ reagent (Gibco/BRL), as per manufacturer's instructions.The transfected cells were selected for 10 days in DMEM+5% FBS(Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants was selected again in 10 μM MTX and 0.5 mg/ml G418 for 10days.

The resulting pool of doubly selected cells was used to generateprotein. Three factories (Nunc, Denmark) of this pool were used togenerate 8 L of serum free conditioned medium. This conditioned mediawas passed over a 1 ml protein-A column and eluted in (10) 750microliter fractions. 4 of these fractions found to have the highestconcentration were pooled and dialyzed (10 kD MW cutoff) against PBS.Finally, the dialyzed material was analyzed by BCA (Pierce) and found tohave a concentration of 317 μg/ml. A total of 951 μg was obtained fromthis 8 L purification.

Example 13 IL-20 Binding Activates STAT3 in the HaCaT Keratinocyte CellLine

IL-20 binds cell lines transfected with both subunits of its receptor.However, these cell lines overexpress the IL-20 receptor relative to itsnormal level and their relevance to the physiological role of IL-20 isunclear. The human HaCaT keratinocyte cell line, which expressesendogenous IL-20RA and IL-20RB was used to examine IL-20 signaltransduction in a biologically relevant cell type. HaCaT cells wereinfected with recombinant adenovirus containing a reporter construct toallow detection of intracellular signaling. The construct consists ofthe firefly luciferase gene driven by promoter/enhancer sequencescomprised of the serum response element (SRE) and signal transducers andactivators of transduction elements (STATs). This assay system detectsproductive ligand-receptor interactions and indicates possibledownstream signal transduction components involved in receptoractivation. Treatment with IL-20 alone resulted in a dose-dependentincrease in luciferase activity with a half maximal response occurringat approximately 2.3 nM. Subsequent luciferase reporter assays usingadenovirus vectors containing only the SRE element or only the STATelements produced detectable reporter activation only through STATs.

To determine if other cytokines act in concert with IL-20, HaCaT cellswere treated with IL-20 alone or in combination with a single submaximaldose of EGF, IL-1β, or TNFα. In the presence of each of these threeproteins, IL-20 treatment resulted in a dose-dependent increase inluciferase activity. IL-20 in combination with IL-1β results in ahalf-maximal response at approximately 0.5 nM, about five-fold lowerthan with IL-20 alone. In addition, activation of the reporter gene isdetectable at 0.1 nM IL-20, a dose that is at least tenfold lower thanthe IL-20 dose required alone.

BHK cells transfected with IL-20RA, IL-20RB or both receptor subunitswere used to determine whether receptor pairing was required for IL-20stimulation of STAT-luciferase. As was the case with binding assays,only cells transfected with both receptor subunits responded to IL-20and did so with a half-maximal response of 5.7 pM. We note that theIL-20 concentration for the half-maximal response in BHK cells is400-fold lower than that for half-maximal response in HaCaT cells. It islikely that a lower concentration of IL-20 is needed for half-maximalresponse in BHK cells, as compared to HaCaT cells, due to higherreceptor levels in the BHK IL-20 receptor transfectants.

A nuclear translocation assay was used to identify STAT proteinsinvolved in IL-20 action. Both HaCaT cells, with endogenous IL-20receptors, and BHK cells transfected with IL-20RA and IL-20RB, weretreated with IL-20 protein and translocation of STAT3 and STAT1transcription factors from the cytoplasm to the nucleus was assayed byimmunofluorescence.

In unstimulated HaCaT cells, STAT3 staining was predominantly in thecytosol. Treatment of HaCaT cells with IL-20 resulted in a distinctaccumulation of STAT3 in the nucleus. Nuclear translocation of STAT3 inresponse to increasing concentrations of IL-20 occurred with ahalf-maximal IL-20 concentration of 7 nM. In contrast to STAT3translocation, HaCaT cells treated with IL-20 did not show anydetectable nuclear accumulation of STAT 1.

BHK cells transfected with IL-20RA and IL-20RB were used to confirm thatthe IL-20 receptor was required for IL-20 stimulation of STAT3 nucleartranslocation. In BHK cells lacking the IL-20 receptor, STAT3 remainedcytosolic following treatment with IL-20. In contrast, in BHK cellstransfected with the IL-20 receptor, STAT3 translocated to the nucleusin response to IL-20. Again, STAT1 remained cytosolic regardless ofIL-20 treatment or IL-20 receptor expression. We conclude that the IL-20receptor is required for IL-20-mediated STAT3 activation.

1. A method of suppressing or reducing IL-20 induced inflammation in amammal suffering from an inflammatory skin disease selected from thegroup consisting of psoriasis, eczema, atopic dermatitis, and contactdermatitis comprising administering to the mammal an effective amount ofa composition comprising an antibody, antibody fragment, or single chainantibody and a pharmaceutically acceptable carrier, wherein theantibody, antibody fragment, or single chain antibody binds to andantagonizes IL-20, and wherein the antibody, antibody fragment, orsingle chain antibody suppresses or reduces inflammation in aninflammatory skin disease selected from the group consisting ofpsoriasis, eczema, atopic dermatitis, and contact dermatitis.
 2. Themethod according to claim 1, wherein the inflammatory skin disease is adefensive response to mechanical trauma, toxins, neoplasia, abnormalrecognition of host tissue as foreign, or invasion of the host byforeign material.
 3. The method according to claim 1, wherein theinflammatory skin disease is an angiogenesis dependent chronicinflammatory condition.
 4. The method according to claim 1, wherein theinflammatory skin disease is a type of psoriasis selected from the groupconsisting of plaque type psoriasis, inverse psoriasis, eruptivepsoriasis, and psoriasis with fingernail involvement.
 5. The methodaccording to claim 1, wherein the composition is administered incombination with another therapeutic agent.
 6. The method according toclaim 5, wherein the therapeutic agent is selected from the groupconsisting of a lubricant, keratolytic, topical corticosteroid, topicalvitamin D derivative, anthralin, systemic antimetabolite, methotrexate,psoralen-ultraviolet-light therapy (PUVA), etretinate, isotretinoin,cyclosporine, and a calcipotriol.
 7. A method of suppressing or reducingIL-20 induced inflammation in a mammal suffering from an inflammatorylung disease selected from the group consisting of bronchitis, lungcancer, bacterial pneumonia, and idiopathic pulmonary fibrosiscomprising administering to the mammal an effective amount of acomposition comprising an antibody, antibody fragment, or single chainantibody and a pharmaceutically acceptable carrier, wherein theantibody, antibody fragment, or single chain antibody binds to andantagonizes IL-20, and wherein the antibody, antibody fragment, orsingle chain antibody suppresses or reduces inflammation in aninflammatory lung disease selected from the group consisting bronchitis,lung cancer, bacterial pneumonia, and idiopathic pulmonary fibrosis. 8.The method according to claim 7, wherein the inflammatory skin diseaseis a defensive response to mechanical trauma, toxins, neoplasia,abnormal recognition of host tissue as foreign, or invasion of the hostby foreign material.
 9. The method according to claim 7, wherein theinflammatory skin disease is an angiogenesis dependent chronicinflammatory condition.
 10. The method according to claim 7, wherein thecomposition is administered in combination with another therapeuticagent.
 11. The method according to claim 10, wherein the therapeuticagent is selected from the group consisting of a lubricant, keratolytic,topical corticosteroid, topical vitamin D derivative, anthralin,systemic antimetabolite, methotrexate, psoralen-ultraviolet-lighttherapy (PUVA), etretinate, isotretinoin, cyclosporine, and acalcipotriol.
 12. A method of suppressing or reducing IL-20 inducedinflammation in a mammal suffering from an inflammatory bowel diseaseselected from the group consisting of ulcerative colitis and Crohn'sdisease comprising administering to the mammal an effective amount of acomposition comprising an antibody, antibody fragment, or single chainantibody and a pharmaceutically acceptable carrier, wherein theantibody, antibody fragment, or single chain antibody binds to andantagonizes IL-20, and wherein the antibody, antibody fragment, orsingle chain antibody suppresses or reduces inflammation in aninflammatory bowel disease selected from the group consisting ofulcerative colitis and Crohn's disease.
 13. The method according toclaim 12, wherein the inflammatory skin disease is a defensive responseto mechanical trauma, toxins, neoplasia, abnormal recognition of hosttissue as foreign, or invasion of the host by foreign material.
 14. Themethod according to claim 12, wherein the composition is administered incombination with another therapeutic agent.
 15. The method according toclaim 14, wherein the therapeutic agent is selected from the groupconsisting of a lubricant, keratolytic, topical corticosteroid, topicalvitamin D derivative, anthralin, systemic antimetabolite, methotrexate,psoralen-ultraviolet-light therapy (PUVA), etretinate, isotretinoin,cyclosporine, and a calcipotriol.
 16. The method according to claim 1,wherein the inflammatory skin disease is psoriasis.